The long-term objective of the proposed research is to determine the role of cultivable and uncultivable oral spirochetes in the etiology of periodontal diseases. there is substantial evidence implicating spirochetes as etiologic agents in periodontal diseases, however, clinical research in this area has been stymied by difficulties in identifying and quantifying these organisms. In acute necrotizing ulcerative gingivitis and some forms of periodontal disease, spirochetes comprise over 50% of the bacterial flora. Currently, there are four known oral spirochete species: Treponema denticola, T. pectinovorum, T. socranskii, and T. vincentii. It is widely believed that there are many more species in the periodontal pocket, but that these species are uncultivable by standard means. DNA probes based upon 16S rRNA sequence information have proven valuable for the rapid identification and quantification or organisms in plaque samples. We have developed DNA probes to the four cultivable oral spirochetes. We have used polymerase chain reaction amplification methods to develop a DNA probe to an uncultivable spirochetes from the digestive tract of oysters. In this proposal, we will use proven methods to develop 16S rRNA based DNA probes to newly identified species of cultivable and uncultivable spirochetes. In Aim 1, culture collections of three collaborating scientists containing 160 oral spirochete strains and freshly isolated strains from our laboratory will be screened using previously developed DNA probes. New species will be identified by 16S rRNA sequence comparative analysis, and from the sequence data we will develop DNA probes that are specific for the new species. In Aim 2, the 16S rRNA sequences for uncultivable spirochetes will be obtained by sequencing cloned DNA from polymerase chain reaction amplification of the 16S rRNA genes from spirochetes in plaque using spirochete specific primers. It is expected that 10 or more new species of spirochetes will be identified in Aims 1 and 2. In Aim 3, pilot clinical studies using in situ hybridization of fluorescently-labeled DNA probes to these oral spirochetes will be performed to determine the prevalence of each spirochete species in gingivitis, acute ulcerative necrotizing gingivitis, and periodontitis. The DNA probes developed in this project will be valuable in future clinical studies to determine the role of specific spirochete species in each of the many types of periodontal diseases. these DNA probes will facilitate studies of spirochete adherence and invasion of epithelial cells, studies of oral ecology and of spirochete pathogenic mechanisms. This project will be of importance to the medical community as well as the dental community.